Diagnostic and therapeutic system for Crohn&#39;s disease and Colitia ulcerosa

ABSTRACT

The present invention relates to a diagnostic system for the detection of Crohn&#39;s disease and ulcerative colitis by means of nucleotide amplification reactions, in situ or in vitro which detection means are specific for the measles virus and are capable of distinguishing between the “wild” type and the attenuated measles virus strains. The invention further provides a use in the manufacture of a medicament for the treatment of Crohn&#39;s disease and/or ulcerative colitis which medicament comprises a suitable vector carrying antisense RNA to specific viral proteins which are consequently inhibited from being expressed in the host.

This application has been filed under 35 U.S.C. 371 from PCT/GB96/00706on Mar. 22, 1996.

The present invention relates to a diagnostic system for the detectionof Crohn's Disease and ulcerative colitis and to a therapeutic systemderived therefrom. No such diagnostic system is currently available.

Until recently there has been no definite cause attributable to theonset of Crohn's Disease or ulcerative colitis which have remainedsubstantially incurable. The best that medicine can provide is somealleviation of the symptoms.

Crohn's Disease afflicts approximately 4 patients per 100,000 of thepopulation, or in the United Kingdom 2,200 new cases per year. Becausein its earlier stages Crohn's Disease tends to present as bowelirritation which is much more common, a reliable diagnostic system isrequired.

Similarly the incidence of ulcerative colitis is sufficient to warrantearly diagnosis to enable early treatment.

In Ann. Med. 1993, Volume 25(6), pages 557 to 561, the inventor and acolleage review the evidence for an association between Crohn's Diseaseand viral infection by various techniques. The inventor and colleaguesalso discusses observations which suggest measles virus is capable ofcausing persistent infection of the intestine, and thus Crohn's Diseasemay be caused by a granulomatous vasculitis in response to the virus inJ. Med. Virol. 1993, Volume 39(4), pages 345 to 353.

Virology 1995, Volume 207(1), pages 168-178 addresses measles virusanti-sense sequences in the treatment of cells persistently infectedwith measles virus.

The applicant has thus shown that the causative agent for Crohn'sDisease is the measles virus and accordingly the 32 present inventionutilises this finding to provide a diagnostic system for the location ofan attenuated vaccine measles virus in bowel tissue, bowel products orin suitable body fluids such as blood or lymph. Continuing studiesincreasingly support the finding that measles virus is the causativeagent for ulcerative colitis. The invention also provides the basis foran anti-viral therapeutic system for the measles virus.

According therefore to a first aspect of the present invention, there isprovided a diagnostic system for the detection of Crohn's Disease andulcerative colitis which system comprises means for detecting attenuatedvaccine measles virus RNA or a distinctive metabolic product thereof.Such means may be an antigenic system, or a system utilizing a nucleicacid amplification or hybridization reaction. In the latter case theinvention specifically may provide means for performing a reversetranscription polymerase chain reaction or a nucleic acid sequence basedamplification reaction or a ligase chain reaction. The systems mayincluding a buffered primer specific for the reverse transcribed DNAfrom a RNA measles virus and/or an RNA template. The primer may comprisea 5′ modified oligonucleotide sequence specific for any measles virusgenomic or antigenomic or messenger RNA. The primer may be attached to alabelling or coagulating moiety such as a fluorochrome for ease ofanalysis.

The distinctive metabolic product may be selected from a gene sequenceor metabolic product thereof to form a nucleocapsid protein, aphosphoprotein, a large protein, an RNA polymerase complex, a matrixprotein, a fusion protein or a haemagglutinin protein, specific for themeasles virus or a related paramyxovirus.

The nucleotide amplification may be a reverse transcription-polymerasechain reaction (RT-PCR) or nucleic acid sequence based amplificationNASBA (or 3SR). In the former case a kit for performing the diagnostictests as hereinbefore set forth may comprise:

(1) M-MLV reverse transcriptase;

(2) random hexamers and/or oligo (Dt)₁₂₋₁₈;

(3) a reaction buffer for (1) above;

(4) a PCR reaction buffer including Taq DNA polymerase

(5) a 5′ modified PCR primer with reporter molecules.

Such a kit may further comprise a positive control which comprisesmeasles virus RNA in solution at a known concentration and preferably anegative control or means for its provision.

We have however now found that NASBA is the preferred diagnostic methodbecause it is significantly more sensitive for the detection of themeasles virus.

According to a further aspect of the present invention there is provideda method for the in situ analysis of a tissue for Crohn's Disease orulcerative colitis which method comprises the steps of:

a) obtaining a tissue sample and securing the same in an enclosedreaction container;

b) adding a reagent comprising a PCR buffer; MgCL₂, dNTP'S, randomhexamers and diluting aqueously to a desired dilution factor;

c) subsequently closing the enclosed reaction container and addingM-MLV-RT and subjecting to heat cycling for at least one cycle;

d) treating with a washing buffer;

e) adding a buffered Taq DNA polymerase, MgCL₂, DNTP and a primer at apredetermined dilution in sterile distilled water and then heat cyclingfor at least 25 cycles; and

f) subsequently repeating step (d) and viewing for labelled product toindicate the presence of a wild or attenuated vaccine measles virus RNA.

In a further aspect of the present invention, there is provided a methodfor the in vitro analysis of a sample for Crohn's Disease or ulcerativecolitis which method comprises the steps of extracting measles virus RNAfrom a tissue sample and

a) adding thereto a buffer further comprising DTT, DNTP'S and R Naseinhibitor and oligo (dT)₁₂₋₁₈ and letting down to a desired aqueousconcentration in an enclosed reaction container;

b) adding thereto M-MLV-RT and subjecting to incubation to provide acDNA product;

c) purifying the product and adding thereto a PCR buffer along withMgCL₂, dNTP's and an outer primer;

d) subsequently adding Taq DNA polymerase and heating for at least 15cycles;

e) recovering an aliquot of a so-formed reaction production and addingbuffered PCR reaction mixture with inner primers and further Taq DNApolymerase and heat cycling for at least 15 cycles; and

f) moving the so-formed product and adding a loading dye and subjectingthe resultant product to an electrophoresis to identify a resultantproduct band;

g) sequencing the amplified products, or hybridising the amplifiedproducts with a homologous or hetrologous specific probe to distinguishvaccine strain measles virus from “wild” type measles virus.

According to a further aspect of the present invention there is provideda medicament for the treatment of induced Crohn's Disease and/orulcerative colitis which method prevents expression, replication,transcription, RNA processing and/or mRNA transport of the measles virusin the host. The medicament may include an attenuated vaccine measlesvirus antisense RNA for either genomic or antigenomic RNA and a vector.Alternatively, the medicament may comprise an attenuated vaccine measlesvirus peptide or carbohydrate antigen or a monoclonal or polyclonalantibody thereof. The invention also comprises a measles virus-specificnucleic acid based vaccine genome encoded into an expression systemadapted to induce a specific immune response to the attenuated vaccinemeasles virus and/or its component antigens.

Using antisense RNA to the viral RNA including messenger RNA for thetreatment of measles associated diseases alleviates the problem of sideeffects on host cellular functions. In the past such treatments haveproved elusive partially due to the fact that it is extremely difficultto design inhibitors specific for the essential proteins of a virus.However, now in the prior art, the antisense RNA approach has beensuccessfully used to inhibit viral genes of avian retrovirus, Roussarcoma virus, human immunodificiency virus and simian immunodifeciencyvirus in cultured cells.

The invention provides a medication which comprises a suitable vectorfor uptake by the infected cells engineered to carry the appropriatemeasles virus antisense RNA designed to be targeted against theappropriate gene (e.g Nucleoprotein gene and/or Haemagglutonin gene)followed by a drug resistance gene (e.g Hygromycin B). The method ofpreparation of the medication is a modification of the methods inKoschel K. et al (1995), Virol. 207, 168-178.

The invention will now be described by way of illustration only in thefollowing examples and with reference

to the attached Figures wherein:

FIGS. 1A-1E shows Measles infected Vero cells immunostained with measlesprimary polyclonal antibody: areas of syncytial cytopathic effectexhibit intense cytoplasmic staining. Counterstained with Mayer'shaematoxylin (original magnification ×350);

FIG. 1b shows Immunogold labelled measles infected Vero cells. Frequentfoci of double and often larger clusters of gold particles are seen,associated with typical fibrillar arrays of nucleocapsids, 15-20 nm indiameter. Counterstained with uranyl acetate and Reynold's lead citrate(original magnification ×91,000);

FIG. 1c shows Measles infected Vero cells treated identically to 1 a,but omitting the measles antibody primary (original magnification ×375);

FIG. 1d shows Measles infected Vero cells developed without measlesprimary antibody, but exposed to gold-conjugated secondary antibody; nolabelling is seen (original magnification ×91,000);

FIG. 1e shows Mumps primary antibody on measles infected Vero cells,showing no signal (immunoperoxidase; original magnification ×400).

FIGS. 2A-2D shows Mumps infected Vero cells immunostained with mumpsprimary antibody showing cytoplasmic staining in infected cells(original magnification ×400);

FIG. 2b shows Mumps infected Vero cells treated identically to 2 a, butomitting the mumps primary antibody (immunoperoxidase; originalmagnification ×400);

FIGS. 2c and 2 d show Measles primary antibody on mumps infected Verocells examined by immunoperoxidase (2 c) (original magnification ×400)and immunogold (2 d) (original magnification ×82,400). No antibody crossreactivity is seen. In 2 d, the fine fibrillary arrays of mumpsnucleocapsids are seen clearly;

FIGS. 3A-3D shows

SSPE: immunostaining for measles virus nucleocapsid protein in SSPEbrain examined by light microscopy (FIG. 3a) original magnification×400) and electron microscopy (FIGS. 3b and 3 c). Intranuclearnucleocapsids are seen in FIG. 3b, frequently showing paired goldparticles spanning individual nucleocapsids, demonstrated clearly inFIG. 3c, showing cytoplasmic signal (original magnifications; 3b×62,500, 3 c×95,000). FIG. 3c, showing cytoplasmic signal (originalagnifications; 3 b×62,500, 3 c×95,000). No signal is seen in identicallyprocessed tissue sections, in which the primary measles antibody wasomitted (FIG. 3d; immunoperoxidase original magnification ×400);

FIGS. 4A-4B shows

Measles appendicitis: immunoperoxidase treated sections stained formeasles virus N protein. Positive signal is seen in an endothelial cellsyncytium (FIG. 4a) (erythrocytes arrowed) (original magnification ×100)and a Warthin Finkeldey giant cell (FIG. 4b) (original magnification×800);

FIGS. 5A-5E shows

Crohn's Disease: positive nuclear staining in macrophage-like cells in afocus of granulomatous inflammation (FIG. 5a; immunoperoxidase; originalmagnification ×600). Signal is not seen in an identically processedserial section, omitting the primary antibody (FIG. 5b, originalmagnification ×600). Positive immunogold labelling is seen in anidentical cellular location and shows the characteristic paired signal(arrowed) FIG. 5c, original magnification ×71,000);

FIG. 5d (inset) shows a high power view of gold label distributed alongthe length of what appears to be a single nucleocapsid (originalmagnification ×139,000);

FIG. 5e shows nuclear staining in a macrophage in a focus ofgranulomatous inflammation from another case of Crohn's Disease. Thepaired signal is observed, particularly in the central cluster, shownmagnified in 5 f; this demonstrates clearly, the parallel arrays(arrowed) of a 17 nm diameter nucleocapsid spanned by the gold label(original magnifications; FIG. 5e×91,000, FIG. 5f×95,000). Staining wasnot observed in otherwise identically processed sections of Crohn'sDisease when the measles primary antibody was omitted (FIG. 5f, originalmagnification ×54,000).

EXAMPLE 1

A study was conducted to confirm the viral origins of thenucleocapsid-like particles observed in bioassays of bowel tissue inCrohn's Disease or probably ulcerative colitis by co-localising theseparticles with a gold-labelled antibody specific for measles virusnucleocapsid protein. These particles have not previously been detectedultrastructurally in normal intestine.

Cell Culture

Vero (green monkey kidney) cells were cultured in 75 mm flasks usingstandard techniques. Confluent monolayers were infected with eitherEdmonston strain measles virus at a titre of 10⁷ plaque forming unitsper ml, or mumps virus (Taylor strain: PHLS Colindale, UK) at a TCID₅₀(Tissue Culture Infectious Dose) of 10⁴; uninfected cells were used as anegative control. When >80% of cells showed cytopathic effect,monolayers were fixed using 1% glutaraldehyde/1% paraformaldehyde inphosphate buffered saline (PBS) for 30 minutes. The cells were scrapedfrom the flask using platinum wire loop and the suspended cells werepelleted in Eppendorf tubes using a Beckman microfuge (Palo Alto,Calif., USA). Cell pellets were washed after fixation with PBS anddehydrated using dimethylformamide, 50, 70 and 90%×2 changes each of10-20 minutes duration depending on the size of the block. The blockswere then infiltrated with a 50:50 mixture of dimethylformamide and LRWhite resin with 0.5% benzoin photoinitiator (TAAB LaboratoriesEquipment Ltd, Reading, UK) added, for 30-60 minutes followed by 100%resin for 1-2 hours. The blocks were embedded in closable embeddingcapsules using fresh LR White with photoinitiator. Polymerisation wascarried out at 40° using a UV light source at 10 cm distance. The resinpolymerised in 1-2 hours and the small amount of resin remainingunpolymerised under the closure was removed sing a cotton swab.

Tissue Reprocessed from Paraffin Blocks

Initially, archival formalin-fixed paraffin processed tissues withestablished measles infection were selected; these included cerebraltissue from a case of subacute sclerosing pan-encephalitis (SSPE), andappendix from a case of acute measles virus appendicitis (Gift of Dr HReid, Chase Farm Hospital, Enfield). Six cases of granulomatous Crohn'sDisease were selected that included 4 rectal biopsies and two appendicestaken at the time of initial presentation (that is, before specific orimmunosuppressive therapy was instituted). In both the positive controlsand Crohn's Disease tissues, the diagnoses were established by standardclinical and histopathological criteria. Two cases of ileocecaltuberculosis (TB) were studied as granulomatous controls. Sections werecut from tissue blocks and immunostained with a polyclonal antibodyspecific for the measles virus nucleocapsid (N) protein (gift of JStephenson and T Fookes, CAMR, Porton Down, UK) using animmunoperoxidase technique, as described previously (Wakefield AJ. J MedVirol 1993; 39;345-353). Serial tissue sections were processed witheither omission of the primary antibody or exposure to mumps (a relatedparamyxovirus) specific monoclonal antibody (Seralab, Crawley, Sussex,UK) as negative controls. Cross-reactivity of the measles and mumpsspecific antibodies was examined in Vero cells infected with theseviruses, prior to immunogold studies.

Processing for Electron Microscopy

Suitable areas of tissue were selected for immunogold analysis; theseincluded areas that gave positive measles staining by immunoperoxidase,and foci of granulomatous inflammation in the cases of Crohn's Diseaseand ileocecal TB. Wedges of tissue were removed from the paraffin blockusing a single edged razor blade. The pieces were dewaxed withchloroform followed by rinsing in absolute alcohol. The tissue was theninfiltrated with resin and embedded as described above.

Paraffin sections only were available on the acute measles appendicitis.A novel method was used to lift the tissue from the slide; the sectionwas dewaxed and taken to absolute alcohol, it was then flooded with50:50 mixture of alcohol and LR White resin with photoinitiator addedfor a further 15 minutes; it was then drained and the slide blotted toremove surplus resin. Drops of fresh resin were placed over the tissueand covered with plastic coverslips (Agar Scientific, Stansted, UK). Theresin was then polymerised with UV at 4° C. for one hour. The coverslipwas peeled off and unpolymerised resin removed. The resin containing thetissue was lifted by immersing the slide in liquid nitrogen for a fewseconds. The pieces were then trimmed and mounted onto blank resinblocks using Permabond quick setting epoxy resin (Permabond AdhesivesLtd, Eastleigh, UK) Ultrathin sections were cut at 50-80 nm andimmunostained as above.

Immunolabelling

Ultrathin section were cut at 70-80 nm and picked up onto Piliform (AgarScientific, Stansted, UK) coated 300 mesh nickel grids (Gilder,Grantham, UK). The grids were incubated on drops of 5% normal goat serumin 0.1% BSA/PBS buffer for 30 minutes, they were then transferred todrops of primary—measles polyclonal antibody 1/100 in 0.1% BSA/PBS for 1hour. Primary antibody was removed by washing on drops of PBS 5×5minutes, the first transfer being made without removing any surplusliquid from the grid. Surplus buffer was removed on the remaining fourchanges by touching the edge on a piece of blotting paper, taking carenot to allow the grid to dry out. The grids were then transferred todrops of gold conjugate (Biocell, Cardiff, UK) diluted 1/100 with PBSfor 1 hour followed by washing with drops of PBS 2×5 minutes to removeunbound gold conjugate and a final rinse in distilled water. Sectionswere stained lightly with uranyl acetate and Reynold's lead citrate andviewed using a Philips 201 transmission electron microscope. Sectionsprocessed omitting the primary antibody, were included with all tissuesas negative controls.

Measles-infected Vero Cells

Vero cells that had been experimentally infected with measles virusexhibited positive immunoperoxidase and immunogold labelling (FIGS. 1aand 1 b) that was not seen either in identically processed measlesinfected cells not exposed to the primary measles antibody (FIGS. 1c and1 d), or in measles infected cells exposed to the mumps primary antibody(FIG. 1e). Ultrastructurally, viral nucleocapsids consisted ofcharacteristic parallel fibrillar structures of between 15 and 20 nmdiameter.

A characteristic pattern of labelling was observed in both nucleus andcytoplasm of infected cells that consisted of gold particles—grouped,often in pairs, and bound at discrete points along the viralnucleocapsids (FIG. 1b); this pattern was seen consistently in bothinfected cells and infected tissues, and was not seen in the veryoccasional background signal observed in control sections. Mumpsinfected Vero cells stained strongly by immunoperoxidase using theprimary mumps antibody (FIG. 2a), but did not stain when the mumpsprimary antibody was omitted in otherwise identically processed cells.No signal was observed when the measles antibody was put onto mumpsinfected cells at either the light microscopic or ultrastructurallevels.

Subacute Sclerosing Panencephalitis

Strong signal for measles virus as observed predominantly in the nucleiof infected cells, using both the immunoperoxidase technique onhistological sections, and immunogold labelling of ultrathin sections ofinfected brain. Ultrastructurally, viral nucleocapsids were wellpreserved despite suboptimal fixation and paraffin processing. Again,the double immunogold signal on nucleocapsids was a consistent feature.No signal was observed on sections in which the primary measles antibodyhad been omitted, either at the light microscopic (FIG. 3d) orultrastructural levels (data not shown).

Intestinal Tissues

The techniques were applied subsequently to measles-infected intestinaltissue—acute infected measles appendicitis—with similar results. Measlesvirus antigen was identified by the immunoperoxidase technique inendothelial cells, and macrophage-like cells including Warthin-Finkeldygiant cells, a classical feature of acute measles virus infection, andoccasional lymphocytes within lymphoid follicles. Staining was not seenin negative control sections. Immunogold labelling confirmed thepresence of the virus in the same cellular foci despite suboptimalpreservation. No signal was observed in the absence of the primaryantibody.

Of the six cases of Crohn's Disease examined, all were positive formeasles by immunoperoxidase within cells consistent in morphology andlocation with histiocytic macrophages, endothelial cells and occasionallymphocytes; signal was not seen in sections that were either notexposed to the measles primary antibody, or exposed to mumps primaryantibody. Five cases were positive by immunogold in the same cellularlocation, and viral particles exhibited the characteristic features ofsize, shape and immunogold labelling described above; in one case,signal was detected additionally, in a single crypt epithelial cell. Inthe fifth case, that was negative by immunogold, the focus ofgranulomatous inflammation present in the histological sections had “cutout” and could not be identified in the ultrathin section. Crohn'ssections, processed in an identical manner, but excluding the measlesprimary antibody, were negative. Of the two cases of ileocecal TB thatwere examined, neither was positive by immunoperoxidase, although onecase exhibited a low level of nuclear signal in a acrophage-like cell,by immunogold labelling.

This presents the first direct evidence of measles virus persistence inthe intestine; that is, the co-localisation of a specific antibody withviral nucleocapsids at the ultrastructural level. The cellularlocalisation of measles virus in Crohn's tissues is consistent with thatobserved previously using different techniques (Wakefield AJ J Med Virol1993; 39; 345-353; Knibbs DR, Gastroenterology, 1993; 104; A726(Abstract)), and in different laboratories (Knibbs DR. Gastroenterology,1993;104;A726 (Abstract)). The co-localisation of a specific antibodywith particles that are, in terms of both size and morphology, identicalto the target virus, is strong evidence for the presence of that virus;the characteristic pattern of labelling of nucleocapsids with goldparticles provided a further, if unexpected degree of specificity. Thedetection of measles in one of two cases of ileocecal TB raises thepossibility that persistently infected immune cells aggregate in foci ofinflammation, and are unrelated to the primary cause of the granuloma.This notwithstanding, in Crohn's Disease tissues, the detection ofmeasles virus within resident intestinal cells, including endotheliumand epithelium, does suggest persistent intestinal infection. It ispossible however that circulating immune cells within the same host mayalso be persistently infected.

Corticosteroids, a mainstay in the treatment of patients with Crohn'sDisease, may predispose to permissive viral replication, and confoundthe question of persistence within the inflamed intestine; none of thepatients whose tissues were examined in this study had received eithercorticosteroids or other immunosuppressive therapy.

EXAMPLE 2

The implied aetiological association of measles virus with Crohn'sDisease is supported by detection of an immune response to infectedcells in affected tissues. This example sought to detect andcharacterise in situ immune responses to measles virus in both acutelyand persistently infected tissues, and in particular, Crohn'sgranulomata. Serial tissue sections from cases of Crohn's Disease(n=17), tuberculosis (n=9), acute intestinal ischaemia (n=5), acutemeasles pneumonitis (n=2), acute measles appendicitis (n=1), subacutesclerosing panencephalitis (SSPE; n=1), and measles inclusion bodyencephalitis (MIBE; n=1), were examined. Single and doubleimmunohistochemical labelling was performed to identify both cytotoxiclymphocytes (CD8, TIA, perforin, Leu 7, CD45RO, CD45RA) and macrophages(KP1). The relationship of these cells to measles infected cells wasidentified by double immunolabelling with anti-measles virusnucleoprotein antibody. In both acute measles appendicitis and SSPE,CD8⁺/TIA⁺ cytotoxic lymphocytes (CTL) targeted infected cells. In theother tissues that were positive for measles virus including Crohn'sDisease (13/17)—where staining was largely confined to granulomata,MIBE, fatal pneumonitis, and 1 tuberculous granuloma, infected cellsappeared to be targeted by macrophages rather CTL. The CTL in Crohn'sgranulomata were Leu 7˜ and perforin˜/CD45RO˜(naive). CTL in bothtuberculous and Crohn's granulomata were similar in their peripheraldistribution, number and phenotype. The data suggest thatmeasles-specific CTL responses may be attenuated in Crohn's Diseasecompared with acute measles appendicitis and SSPE, and secondly, that anabnormal macrophage response to persistent measles virus infection ofthe intestine could result in granulomatous inflammation.

EXAMPLE 3

In an international collaborative study, serum measles IgMimmunoreactivity was assayed by ELISA in consecutive outpatients withCrohn's Disease (n=95), ulcerative colitis (n=79), viral hepatitis(n=63) and blood donors (n=30). Results were compared with thoseobtained from a different commercial measles IgM assay, serum rubellaand Epstein-Barr virus-specific IgM immunoreactivity, total serum IgM,Rheumatoid Factor and measles-specific IgG. Twenty patients withinflammatory bowel disease were studied serially over a four monthperiod. At the ELISA cut-off point for confirmation of recent acutemeasles virus infection, there were no significant differences betweengroups. However, increased serum measles IgM immunoreactivity (≧mean±2SDof blood donors) was significantly greater in patients with Crohn'sDisease 39/95 (41%) and ulcerative colitis 33/79 (42%) compared withhepatitis patients 5/64 (8%) and normal controls 0/30 (0%) (p<0.001).Those positive by ELISA were also positive by indirectimmunofluorescence on the same serum sample. Serum measles IgMimmunoreactivity did not correlate with either total IgM, rubella orEpstein-Barr virus IgM (not raised), measles IgG, or disease activity.Patients not receiving steroids were more likely to have raised measlesIgM immuunoreactivity (p<0.05). All sera examined for Rheumatoid Factorwere negative. Of twenty patients with inflammatory bowel diseasestudied over four months, 55% showed raised measles IgM immunoreactivityat some stage during the follow-up. The data suggested an immunologicalresponse to measles virus in patients with Crohn's Disease andulcerative colitis, supporting a potential aetiological role for thisagent in inflammatory bowel disease.

EXAMPLE 4

The epidemiological association between Crohn's Disease and earlymeasles virus exposure has been indicated either indirectly, on instudies of case-control design. In order to determine absolute riskestimates for in utero measles exposure and Crohn's Disease, maternitycharts for all 25,000 deliveries at University Hospital, Uppsala,between 1940-1949 were reviewed; four cases of overt measles infectionin the mother during pregnancy were identified. Offspring, and in twocases their mothers also, were interviewed, and case records reviewed.Cases, 1, 2 and 3 had undergone multiple intestinal resections; tissues,available from two cases, were examined by both routine histology, andimmunohistochemistry and immunogold electronmicroscopy for measles.virus, using appropriate positive (brain; subacute sclerosingpanencephalitis) and negative (no primary antibody, mumps infectedcells) controls (Gut. 1995; 36: 564-9). Three cases of Crohn's Diseaseoccurred in the four offspring; in each case the disease was preceded byrecurrent, antibiotic resistant pneumonia. All had extensive ileal andcolonic disease, two patients requiring intravenous feeding. Of the fourcases, the only one to have suffered clinical acute measles disease, didnot develop Crohn's Disease. Tissues from two cases of Crohn's Diseasethat were examined for measles virus antigen, were positive in foci ofgranulomatous and lymphocytic inflammation. The data indicate that inutero exposure to measles virus is a major risk factor for severe,extensive Crohn's Disease. Exposure at this time may lead to persistentinfection, or alternatively, modify the response to the infection inlater life, leading to virus persistence.

EXAMPLE 5

Immunogold electron microscopy was used to test for the presence ofmeasles virus in intestinal tissue from patients with Crohn's Disease,and inflammatory and non-inflammatory disease controls. Formalin-fixed,paraffin-embedded tissue was reprocessed and stained with anti-measlesnucleoprotein primary antibody followed by 10 nm gold-conjugatedsecondary antibody. Similarly processed brain tissue, taken from apatient with subacute sclerosing panencephalitis (SSPE), was used as thepositive control. Duplicate sections of all tissues were processedwithout the primary antibody. In Crohn's Disease 8/9 foci ofgranulomatous inflammation and 0/4 foci of non-specific inflammationwere positive for measles virus. Of control,s 0/5 ulcerative colitistissues and 1/1 SSPE tissues were positive. Gold grain counts pernuclear field in both Crohn's granulomata and SSPE were highlysignificantly greater than controls, including non-granulomatous areasof Crohn's Disease (p<0.0006); in both diseases staining was confined toa small population of cells exhibiting characteristic cytopathology.These data support a role for measles virus in the aetiology of Crohn'sDisease.

EXAMPLE 6

Kits for Measles Virus Detection

Measles is an RNA virus. The nucleic acid amplification mediateddetection whether in situ or in vitro of this example is based on eitherreverse transcription—polymerase chain reaction (RT-PCR) or nucleic acidsequence based amplification NASBA (3SR).

The main components of the RT-PCR kit for measles detection in vi tro orin situ possess

(i) Reverse transcriptase eg M-MLV reverse transcriptase

(ii) Primers for first strand cDNA synthesis

iii) reaction buffer for RT,

(iv) PCR reaction buffer,

(v) PCR primers which may be 5′ modified with reporter molecules, egfluorochromes.

Additionally an Amicon Microcon 30 size exclusion cartridge isoptionally required for purification of the product from first strandDNA synthesis for in vitro amplifications.

Both kits preferably include a positive control, for example measlesvirus RNA, supplied as a solution at known concentration with whichindividuals using the kit can calibrate their results.

Additionally a negative control is required. This is pref erablyachieved by treatment of one portion of the sample to be tested withRNase A for 10 minutes at 37° C. prior to analysis. This is effectivefor both in situ and in vitro applications and additionally indicatesany non-specific product formation from any DNA that may be present as acontaminant in the samples. It is therefore desirable to include Rnase Aas an additional component in the kit.

Two basic protocols follow for a) in situ; and b) in vitroamplification.

EXAMPLE 7

a) Measles virus detection in situ—Kit 1

1) To the tissue section as prepared in Example 6 is added 75 μl ofreagent (1). The slide is sealed in an enclosed reaction chamber such asthat shown in PCT/GB95/00215 and placed on a flat block of a pHC3 Technethermal cycler. With the enclosed reaction container so positioned 200units of M-MLV RT are added and heated for 2 hours at 42° C., followedby five minutes at 95° C., followed by five minutes at 15° C. tocomplete a first heating cycle.

2) The slide is then thoroughly rinsed in the reagent (2), and

3) Subsequently the tissue section is covered with 430 μl of the reagent(3). To this is added 1.25 units of Taq DNA polymerase and the whole isheated for five minutes at 95° C. followed by thirty heat cycles of twominutes at 58° C., 1.5 minutes at 75° C. and 1 minute at 95° C. Theaction is finished by heating for ten minutes at 72° C.

4) The slide is then rinsed thoroughly in reagent (2) and viewed underan epifluoresence microscope.

Reagents for Kit 1 in situ measles detection

Reagent 1 (RT buffer—inc.primers)

q

10×PCR buffer

MgCL₂

dNTPs

RNA Guard

Random Hexamers

DEPC treated water

At required final

working concentration

Reagent 2 (Washing buffer)

phosphate buffered saline Ph 7.5

Reagent 3 (PCR buffer—inc.primers)

10×PCR buffer

MGCL₂

DNTPS

Primer 1) either 5′ modified

Primer 2) or +Dig-UTP

Sterile double distilled water

At required final

working concentration

Enzymes

M-MLV RT—(Eg Gibco BRL)

Taq DNA polymerase—(eg Gibco BRL)

EXAMPLE 8

b) Measles nucleic acid detection bv amplification in vitro Kit 2

It is assumed that total RNA will have been prepared from tissues ofinterest for example by the method of Chirgwin et al (1979)Biochemistry, 18, 5294-5299, the diagnostic test may then be effected bythe following steps:

1) To 0.4 μg of RNA add sufficient 5×reagent (4) 1 and DEPC treatedsterile double distilled water to make up volume to 20 μl.

2) Remove an aliquot (containing 100 ng) of total RNA for an internalcontrol reverse transcriptive PCR using the low copy number cellular UIARNA as a target for amplification;

3) Perform hybrid capture on UIA positive RNA samples using magneticbeads with amino-linked oligonucleotide specific for both positive andnegative measles RNA strands;

4) Separate the magnetic beads and elute measles RNA with 50 μl ofelution buffer;

5) Use 5 μl of eluent for measles reverse transcription PCR togetherwith relevant positive and negative controls.

6) Add 200 units of M-MLV RT and incubate at 42° C. for 2 hours for 30minutes

7) Take the resultant cDNA product and purify using an Amicon Microcon30 cartridge according to the manufacturers instructions.

8) Take 10 μl of purified cDNA product from step 3 and make up to 39 μlwith a total of 10×reagent (5) and sterile double distilled water.

9) Add 1 unit of Taq DNA polymerase and heat for 10 minutes at 95° C.followed by 35 heat cycles of 58° C. for one minute, 72° C. for 1 minuteand 95° C. for 1 minute. Finished with one heat cycle of 72° C. for 5minutes

10) Take 1 μl of the reaction product from step 5 and make up to 23 μlwith 10×reagent (6) and sterile double distilled water.

11) Add 1 unit of Taq DNA polymerase and heat cycle as for step 5 butfor 25 rather than 35 cycles.

12) Take 18 μl of product from step 7 add 2 μl of loading dye andelectrophrese on a 1% agarose gel to identify product of a desired kbp.

Reaqents for Kit 2 in vitro measles detection

Reagent 5 Reagent 6 Reagents 4 (PCR buffer 1) - (PCR buffer 2) - (RTbuffer) inc primers inc primers RT buffer PCR buffer PCR buffer DTTMgCL₂ MgCL₂ dNTPs W − 1 W − 1 RNase inhibitor dNTPs dNTPs Oligo(dT)₁₂₋₁₈ MV1) outer primers inner primers MV2) MV4) At 5 × working At10 × working At 10 × working concentration concentration concentration

Enzymes

M-MLV RT—(eg Gibco BRL)

Taq DNA polymerase—(eg Gibco BRL)

Additionally

DEPC treated sterile double distilled water

Amicon Microcon 30 size exclusion cartridge.

EXAMPLE 9

NASBA Amplification of Measles Virus N Gene Sequence

1. Warm NASBA buffer and primer, mix at room temperature and make upmaster mix for 20 μl reactions in clean room.

2. Aliquot master mix into Eppendorf tubes and add 100 ng of templateRNA per reaction prepared by the method of Cosby et al 1989.

3. Maintain tubes at 65° C. for 5 minutes to denature RNA then hold at41° C. for 5 minutes.

4. Warm enzyme mix to room temperature then add to reaction mix,

5. Incubate at 41° C. for 90 minutes in a water bath.

6. Detect reaction products via Northern blotting or use anenzyme-linked gel assay (e.g ELGA, Organon or Technika).

NASBA BUFFER VOLUME FINAL REAGENT (μl) CONCENTRATION 1M Tris pH 8.5 20040 mM 1M MgCl₂ 60 12 mM 4M KCl 87.5 70 mM 1M DTT 25 5 mM 20 mM each dNTP50 each 1 mM each 20 mM ATP, UTP, CTP 100 each 2 mM each 20 mM GTP 751.5 mM 100 mM ITP 25 0.5 mM H₂O 27.5 1000 μl

Aliquot and stored at −20° C.

PRIMER MIX VOLUME FINAL REAGENT (μl) CONCENTRATION 100% DMSO 750 75% 50uM Primer 1 20 1 uM 50 uM Primer 2 20 1 uM H₂O 1000 μl

Aliquot and store at −20° C.

ENZYME MIX VOLUME FINAL REAGENT (μl) CONCENTRATION H₂O 969 1.5 M  4.5 MSorbitol 668 105 ng/μl   20 mg/ml BSA 42 105 ng/μl 0.87 U/μl Rnase 11 360.004 U/μl   70 U/μl T7 RNA Pol. 184 1.6 U/μl 25.3 U/μl AMV RT 101 0.32U/μl 2000 μl

Aliquot and store at −70° C.

NB. Do not vortex the enzyme mix.

EXAMPLE 10

Measles RNA extraction and detection using NASBA was effected using thefollowing steps:

1. Homogenise up to 500 mg of tissue in 3 ml total RNA isolation reagent(Advanced Biotechnologies). Total RNA extraction method is based on thesingle-step method of RNA isolation by Chomczynski et al. If usingcells, resuspend pellet in 1 ml of reagent. If using blood, mix with5×vol. of reagent.

2. Extract total RNA and resuspend pellet in 100 μl of DEPC treateddH₂O.

3. Measure optical density of extracted RNA solution and determine theRNA integrity via gel electrophoresis on a 1.5% agarose gel.

4. Remove an aliquot (containing 100 ng) of total RNA for an internalcontrol NASBA using the low copy number cellular U1A RNA as a target foramplification.

5. Perform hybrid capture on UIA positive RNA samples using magneticbeads coated with amino-linked oligonucleotides specific for bothpositive and negative measles RNA strands.

6. Separate the magnetic beads and elute measles RNA with 50 μl ofelution buffer.

7. Use 5 μl of eluent for measles NASBA together with relevant positiveand negative controls.

Detect specific NASBA products using Enzyme Linked Gel Electrophoresiswith an internal horseradish peroxidase-labelled oligonucleotide.

Measles NASBA

Measles NASBA should be performed using primers specific to thenucleoprotein region of the measles virus genome for example, primersAB20 and AB22.

Specific NASBA products should be detected using an internal oligo probefor example AB20.

OLIGO POSITION SEQUENCE AB20 (NASBA) 1200-1219 AGG GCA AGA GAT GGT AAGGA (Upstream) AB22 (NASBA) 1358-1379 AAT TCT AAT ACG ACT CAC TAT(Downstream) AGG GGA TCA CCG TGT AGA AAT GAC A AB10 (PROBE) 1288-1308GTT TCA GAG ATT GCA ATG CA

EXAMPLE 11

In situ Hybridisation Detection of Measles Virus

It is possible to detect measles virus RNA in situ in tissue sectionswhich have been , for example, paraffin or araldite embedded or frozen,via in situ hybridisation.

Detection of hybrids can be either direct i.e. by fluorescence orautoradiography or indirect i.e. by subsequent reaction of the hybridwith a reporter molecule to allow its detection, for example, bychemiluminescence or fluorescence.

The following example method is for the identification of measles virusRNA in tissue sections employing a biotinylated single stranded RNAprobe and the subsequent immuno-detection of hybrids. This probe isdevised from the N gene sequence (Cosby et al 1989) and is 186 bp inlength. It is specific for all nucleocapsid sequences of measles viruscontained in the GenBank sequence data base but will not react with theclosely related morbillivirus—canine distemper virus.

HYBRIDISATION

1. Take either semi-thin or ultra-thin sections and, if paraffinembedded, dewax and rehydrate (on slide), add 200 μl of proteinase K (1mg ml⁻¹), and incubate for 15 ml at 37° C.

2. Wash in DEPC treated water and paraformaldehyde fix for 5 min.

3. Wash sections in DEPC treated water.

4. Add hybridisation buffer and incubate at 42° C. for 16 hours.

5. Wash sections in 0.6% NaCl, 10 mM HCl pH 7.0, Immol EDTA for 5minutes at 25° C.

6. Rinse sections in DEPC treated water.

7. Wash sections in 45% v/v formamide in washing buffer for 30 minutesat 28° C.

8. Rinse sections in DEPC treated water.

9. Wash sections in 0.1×SSC for 60 minutes at 40° C.

10. Wash sections in lommol phosphate buffered saline twice for 5minutes each at 25° C.

Solution

Hybridisation buffer (Final Concentration)

Formamide (50% v/v)

b 5×SSC

5×Denhardts solution

0.25 mg ml⁻¹ Salmon Sperm DNA

0.5 mg ml⁻¹ yeast tRNA

10% Dextran sulphate

DEPC treated water

Probe RNA≦100 nmol

DETECTION (adapted from Cosby et al 1989 and McQuaid et al 1990)

1. Monoclonal mouse anti-biotin antibody at 1:40 dilution is applied at37° C. for 30 minutes to the section.

2. Wash sections twice in excess phosphate buffered saline.

3. Add biotinylated anti-mouse antibody at a 1:90 dilution and incubatefor 30 minutes (ibid) at 37° C.

4. Wash sections twice in excess phosphate buffered saline.

5. Add a 1:500 dilution of peroxidase conjugate and incubate for 30 minat 25° C.

6. Rinse the sections in excess phosphate buffered saline for 8 minutes

7. Add substrate 3-amino-9-diethylcarbazole and leave at 25° C. for 10minutes.

8. Wash sections in running water for 10 minutes.

9. Allow to dry and view under microscope.

10. Sections can be counter stained with Mayers haematoxylin ifappropriate.

A DETECTION KIT using the above technique comprises:

(a) the riboprobe;

(b) hybridisation buffer;

(c) proteinase K;

(d) Rnase A; and

(e) antibodies and detection reagents.

A positive and negative control are included where the negative controlmay be generated by the pre-treatment of an arbitrary, duplicatedsample, with RNase A for 1 hour at 37° C. prior to hybridisation anddetection. A positive control for the kit may be a sample of plasmidborne transcription template and for section detection the control couldbe a sample of a known SSPE infected tissue.

The kits in accordance with the present invention locate measles RNAwhich is indicative of Crohn's Disease from tissue samples from biopsiesof the bowel and from body fluids such as blood and lymph and fromfaecal extracts.

The invention relates therefore to a diagnostic system for the detectionof Crohn's Disease and ulcerative colitis to assay kits for utilisationof said system, and to therapeutic systems derived therefrom.

What is claimed is:
 1. A diagnostic method for the detection of Crohn'sDisease and/or ulcerative colitis which comprises detecting measlesvirus RNA in granulomas and/or lymphoid follicles in bowel tissue. 2.The diagnostic method according to claim 1 wherein virus RNA is detectedby performing a nucleic acid based test in vitro.
 3. The diagnosticmethod according to claim 2 wherein the test comprises a nucleotideamplification or hybridization reaction selected from a polymerase chainreaction (RT-PCR) or a nucleic acid based amplification reaction(NASBA).
 4. The diagnostic method according to claim 3 wherein the testcomprises the steps of: (a) extracting measles RNA and detecting thesame using NASBA or RT-PCR; (b) enriching specific measles virusRNA/mRNA; and (c) repeating NASBA and RT-PCR.
 5. The diagnostic methodaccording to claim 2 wherein a buffered primer comprising a 51′ modifiedoligonucleotide sequence specific for the reverse transcribed DNA froman RNA measles virus is used to detect the virus RNA.
 6. The diagnosticmethod according to claim 3 wherein the test comprises a RNA templatespecific for a RNA measles virus.
 7. The diagnostic method according toclaim 2 wherein a primer or probe is attached to a reporter moleculewhich enables post amplification detection of the virus RNA.
 8. Thediagnostic method according to claim 7 wherein the reporter molecule isflurochromatic.
 9. The diagnostic method according to claim 5 furthercomprising use of the following reagents: (1) M-MLV-reversetranscriptase; (2) random hexamers and/or oligo (dT)₁₂₋₁₈; (3) areaction buffer for (1) above; (4) a PCR reaction buffer; and (5) a 5′modified PCR primer or probe with reporter molecules.
 10. The diagnosticmethod according to claim 9 further comprising use of a positive controlcomprising measles virus RNA in solution at a known concentration and anegative control or means for providing same.
 11. The diagnostic methodaccording to claim 1 wherein the measles virus RNA is detected via adistinctive metabolic product selected from a gene sequence or ametabolic product thereof to form a nucleocapsid protein, aphosphoprotein, a matrix protein, a RNA polymerase complex, ahemagglutinin protein, a fusion protein and/or another characteristicproduct thereof detectable by an immunological method.
 12. Thediagnostic method according to claim 11 wherein the distinctivemetabolic product is obtained from a sample of bowel tissue containinggranulomas or lymphoid follicles.
 13. A method for the analysis ofCrohn's Disease and/or ulcerative colitis which method comprises thesteps of: (a) obtaining a sample of bowel tissue containing granulomasor lymphoid follicles from a patient, and securing the same in anenclosed reaction container; (b) adding a reagent comprising a PCRbuffer, MgCl₂ and DNTP random hexamers diluted aqueously to a desireddilution factor; (c) subsequently closing the enclosed reactioncontainer, adding M-MLV-RT and subjecting to heat cycling for at leastone cycle; (d) treating with a washing buffer; (e) adding a buffered TaqDNA polymerase, MgCl₂, DNTP and a base primer or probe at apredetermined dilution and then heat cycling for at least 25 cycles toproduce a labeled product; (f) subsequently repeating step (d) andviewing for the labeled product to indicate the presence of measlesvirus RNA; and (g) determining whether the patient suffers from Crohn'sDisease or ulcerative colitis based upon the presence or absence of themeasles virus RNA.
 14. A method according to claim 13 wherein the baseprimer or probe is a Dig-11 or fluorescent labeled base primer or probe.15. A method for the in vitro analysis of a sample of bowel tissuecontaining granulomas or lymphoid follicles from a patient with Crohn'sDisease and/or ulcerative colitis which method comprises abstractingmeasles virus RNA from bowel tissue containing granulomas or lymphoidfollicles and: (a) adding thereto a buffer further comprising DTT, DNTPsand RNase inhibitor and oligo (Dt) 12-18; (b) adding thereto M-MLV-RTand subjecting to incubation to provide a cDNA product; (c) purifyingthe product and adding thereto a PCR buffer along with MgCl₂, dNTPs andan outer primer or probe; (d) subsequently adding Taq DNA polymerase andheating for at least 15 cycles, whereby a first reaction product isformed; (e) recovering an aliquot of the first reaction product andadding buffered PCR with inner primers or probes and further Taq DNApolymerase and recycling for at least 15 cycles, whereby a secondreaction product is formed; (f) removing the second reaction product andadding a loading dye to form a dyed product and subjecting the dyedproduct to electrophoresis to identify a selected product band; and (g)sequencing the amplified products or hybridizing the amplified productswith a homologous or heterologous specific probe to distinguish themeasles virus to abstract the measles virus RNA from the tissue.
 16. Amethod for the in vitro analysis of a sample of bowel tissue containinggranulomas or lymphoid follicles from a patient with Crohn's Diseaseand/or ulcerative colitis which method comprises abstracting measlesvirus RNA from bowel tissue containing granulomas or lymphoid folliclesand: (a) adding thereto a room temperature buffer further comprisingDNTP and room temperature primer or probe mixes to produce a master mix;(b) aliquoting master mix into a reaction mixture comprising thetemplate RNA to produce a resulting mixture, heating the resultingmixture for 5 minutes at 65° C., then at 41° C. for 5 minutes; (c)subsequently adding the enzyme mix further comprising a RNA polymerase;(d) incubating at 41° C. for 90 minutes in a water bath to produce areaction product; (e) detecting the reaction product by Northernblotting or an enzyme-linked gel assay; and (f) sequencing of theamplified products or hybridizing the amplified products with ahomologous or heterologous specific probe to distinguish the measlesvirus.
 17. A diagnostic method for the detection of Crohn's Diseaseand/or ulcerative colitis comprising detecting measles virus or viralparticles in granulomas and/or lymphoid follicles in bowel tissuewherein the presence of said virus or particles is diagnostic of Crohn'sDisease and/or ulcerative colitis.